Bowen Dempsey

Mapping and analysis of the connectome of sympathetic premotor neurons in the rostral ventrolateral medulla of the rat using a volumetric brain atlas

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) play a critical role in the generation of vasomotor sympathetic tone and are thought to receive convergent input from neurons at every level of the neuraxis; the factors that determine their ongoing activity remain unresolved. In this study we use a genetically restricted viral tracing strategy to definitively map their spatially diffuse connectome. We infected bulbospinal RVLM neurons with a recombinant rabies variant that drives reporter expression in monosynaptically connected input neurons and mapped their distribution using an MRI-based volumetric atlas and a novel image alignment and visualization tool that efficiently translates the positions of neurons captured in conventional photomicrographs to Cartesian coordinates. We identified prominent inputs from well-established neurohumoral and viscero-sympathetic sensory actuators, medullary autonomic and respiratory subnuclei, and supramedullary autonomic nuclei. The majority of inputs lay within the brainstem (88–94%), and included putative respiratory neurons in the pre-Bötzinger Complex and post-inspiratory complex that are therefore likely to underlie respiratory-sympathetic coupling. We also discovered a substantial and previously unrecognized input from the region immediately ventral to nucleus prepositus hypoglossi. In contrast, RVLM sympathetic premotor neurons were only sparsely innervated by suprapontine structures including the paraventricular nucleus, lateral hypothalamus, periaqueductal gray, and superior colliculus, and we found almost no evidence of direct inputs from the cortex or amygdala. Our approach can be used to quantify, standardize and share complete neuroanatomical datasets, and therefore provides researchers with a platform for presentation, analysis and independent reanalysis of connectomic data.

Excessive Respiratory Modulation of Blood Pressure Triggers Hypertension

The etiology of hypertension, the worlds biggest killer, remains poorly understood, with treatments targeting the established symptom, not the cause. The development of hypertension involves increased sympathetic nerve activity that, in experimental hypertension, may be driven by excessive respiratory modulation. Using selective viral and cell lesion techniques, we identify adrenergic C1 neurons in the medulla oblongata as critical for respiratory-sympathetic entrainment and the development of experimental hypertension. We also show that a cohort of young, normotensive humans, selected for an exaggerated blood pressure response to exercise and thus increased hypertension risk, has enhanced respiratory-related blood pressure fluctuations. These studies pinpoint a specific neuronal target for ameliorating excessive sympathetic activity during the developmental phase of hypertension and identify a group of pre-hypertensive subjects that would benefit from targeting these cells.

Recording, labeling, and transfection of single neurons in deep brain structures

Genetic tools that permit functional or connectomic analysis of neuronal circuits are rapidly transforming neuroscience. The key to deployment of such tools is selective transfection of target neurons, but to date this has largely been achieved using transgenic animals or viral vectors that transduce subpopulations of cells chosen according to anatomical rather than functional criteria. Here, we combine single‐cell transfection with conventional electrophysiological recording techniques, resulting in three novel protocols that can be used for reliable delivery of conventional dyes or genetic material in vitro and in vivo. We report that techniques based on single cell electroporation yield reproducible transfection in vitro, and offer a simple, rapid and reliable alternative to established dye‐labeling techniques in vivo, but are incompatible with targeted transfection in deep brain structures. In contrast, we show that intracellular electrophoresis of plasmid DNA transfects brainstem neurons recorded up to 9 mm deep in the anesthetized rat. The protocols presented here require minimal, if any, modification to recording hardware, take seconds to deploy, and yield high recovery rates in vitro (dye labeling: 89%, plasmid transfection: 49%) and in vivo (dye labeling: 66%, plasmid transfection: 27%). They offer improved simplicity compared to the juxtacellular labeling technique and for the first time offer genetic manipulation of functionally characterized neurons in previously inaccessible brain regions.

The connectome of rostral ventrolateral medulla sympathetic premotor neurons

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) are thought to play a critical role in the generation of vasomotor sympathetic tone and represent a major site of convergence for multiple descending and reflex pathways that co-ordinate sympathetic nerve activity. Elucidating the organization of the circuits that drive these neurons is a key research objective. Here we present brain-wide connectomic maps of neurons that provide monosynaptic drive to putative RVLM sympathetic premotor neurons, generated using a two-step restricted trans-synaptic viral tracing strategy. We made focal microinjections of a genetically restricted reporter-expressing rabies vector, SADΔG(EnvA)-RFP, into the RVLM, and restricted its entry to neurons that project to the T2 spinal cord by first transducing them with a cassette required for rabies entry and trans-synapsis, YTB. By precisely targeting rabies injections to the core of the RVLM bulbospinal population we were able to restrict rabies ‘seeding’ to small numbers (as low as one single neuron) of RVLM presympathetic neurons immediately caudal to the facial nucleus, and then map the locations of neurons that provide monosynaptic input to the seed population. We observed reproducible patterns of inputs arising from the dorsal, contralateral, and midline medulla, and local RVLM interneurons including catecholaminergic non-bulbospinal neurons and neurons likely to reside within the ventral respiratory column. Distant inputs were identified in the pons, cerebellum and midbrain, and included previously suspected sites of monosynaptic drive such as the paraventricular nucleus of the hypothalamus.